Objective： N-acetylneuraminic acid is widely present in human breast milk and animal milk. This study intends to establish a method for the determination of N-acetylneuraminic acid in different milks. Methods： The high-performance anion exchange chromatography coupled with pulsed amperometric detector method （HPAEC-PAD） was used to establish and verify a method for determining the content of total N-acetylneuraminic acid in breast milk and cow milk. Results： The method has simple pretreatment， excellent linearity in a wide linear range （20.7-4 000 mg/L）， with a correlation coefficient greater than 0.999， and the limits of detection and quantification are 4.3 mg/L and 20.7 mg/L， respectively. The linear range and limit of quantification of the method were satisfied the need to determinate the content of N-acetylneuraminic acid in breast milk over 1 years and commercially available cow milk. The method is applicable to the determination of N-acetylneuraminic acid in different milks. The accuracy and precision of the method are good， and the recovery of spike addition is between 86.1%-99.3%. The RSD of six repeated measurements is less than 1.9%. The content of total N-acetylneuraminic acid in breast milk within a year after delivery is 187.1-1334.7 mg/L， and the content shows a downward trend along lactation period. The proportion of free N-acetylneuraminic acid in breast milk is 1.0%-2.5%. The content of N-acetylneuraminic acid in five kinds of commercially available cow's milk is 177.6-228.9 mg/L of which free N-acetylneuraminic acid accounted for 8.5%-11.2%. Conclusion： This method is suitable for rapid and accurate determination of total N-acetylneuraminic acid in breast milk and cow milk.